IIn this EUREKA application, entitled "Split Proteins As Boolean Circuits and Drugs of a New Kind", we propose to verify, optimize, and implement a new approach to therapy of "complex" diseases. In this approach, termed deletion-specific targeting (DST), the abnormal cells are targeted, in a novel way, through mutational changes in their DNA, specifically homozygous DNA deletions (HDs). For example, many cancers have been shown to harbor HDs. (Although cancer is a relevant example, the same "DST" logic applies to any undesirable cell lineage that can be shown to contain HDs. In other words, the DST strategy is also relevant to diseases other than cancer.) A salient property of a homozygous deletion is that this "negative" target cannot revert, thereby serving as an immutable signpost for therapy. To target an HD, i.e., a "target" that is absent, the DST strategy brings together, in a novel way, both existing and new methodologies, including the ubiquitin fusion technique, split-ubiquitin assay, zinc-finger DNA-recognizing proteins and split restriction meganucleases. The DST strategy also employs a feedback mechanism that receives input from a circuit operating as a Boolean OR gate and involves the activation of split meganucleases, which destroy DST vector in normal (non-target) cells. The logic of DST makes possible an essentially unlimited increase in the selectivity of therapy. We propose to test and develop DST initially in the yeast S. cerevisiae, a genetically tractable organism in which sophisticated manipulations are faster and easier than in mammalian cells. This stage of the project will be followed by tests of resulting (optimized) circuits in mammalian cells in culture, and thereafter in intact mice with specific diseases. Project Narrative: The studies proposed in this EUREKA application are designed to verify, develop and implement a new approach to therapy of "complex" diseases. This approach, termed deletion-specific targeting (DST), makes possible the targeting of homozygous deletions (HDs) in abnormal cells, e.g., in cancer (but not only cancer) cells. The eventual implementation of DST strategy in a clinical setting may prove to be curative with a range of HD-relevant diseases, and in addition substantially free of side effects.